Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana
Fristedt, Rikard ; Hu, Chen ; Wheatley, Nicole ; Roy, Laura M. ; Wachter, Rebekka M. ; Savage, Linda ; Harbinson, Jeremy ; Kramer, David M. ; Merchant, Sabeeha S. ; Yeates, Todd ; Croce, Roberta - \ 2018
The Plant Journal 94 (2018)1. - ISSN 0960-7412 - p. 146 - 156.
Abscisic acid - Atg5 g51110 - Chloroplast - RAF2 - RuBisCO - RuBisCO aggregation - RuBisCO assembly factor - SDIRIP1
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO2) and ribulose-1,5-bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD-like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.
Turn-taking in cooperative offspring care : by-product of individual provisioning behavior or active response rule?
Savage, James L. ; Browning, Lucy E. ; Manica, Andrea ; Russell, Andrew F. ; Johnstone, Rufus A. - \ 2017
Behavioral Ecology and Sociobiology 71 (2017)11. - ISSN 0340-5443
Cooperative breeding - Parental care - Provisioning rules - Reciprocity
Abstract: For individuals collaborating to rear offspring, effective organization of resource delivery is difficult because each carer benefits when the others provide a greater share of the total investment required. When investment is provided in discrete events, one possible solution is to adopt a turn-taking strategy whereby each individual reduces its contribution rate after investing, only increasing its rate again once another carer contributes. To test whether turn-taking occurs in a natural cooperative care system, here we use a continuous time Markov model to deduce the provisioning behavior of the chestnut-crowned babbler (Pomatostomus ruficeps), a cooperatively breeding Australian bird with variable number of carers. Our analysis suggests that turn-taking occurs across a range of group sizes (2–6), with individual birds being more likely to visit following other individuals than to make repeat visits. We show using a randomization test that some of this apparent turn-taking arises as a by-product of the distribution of individual inter-visit intervals (“passive” turn-taking) but that individuals also respond actively to the investment of others over and above this effect (“active” turn-taking). We conclude that turn-taking in babblers is a consequence of both their individual provisioning behavior and deliberate response rules, with the former effect arising through a minimum interval required to forage and travel to and from the nest. Our results reinforce the importance of considering fine-scale investment dynamics when studying parental care and suggest that behavioral rules such as turn-taking may be more common than previously thought. Significance statement: Caring for offspring is a crucial stage in the life histories of many animals and often involves conflict as each carer typically benefits when others contribute a greater share of the work required. One way to resolve this conflict is to monitor when other carers contribute and adopt a simple “turn-taking” rule to ensure fairness, but natural parental care has rarely been studied in sufficient detail to identify such rules. Our study investigates whether cooperatively breeding chestnut-crowned babblers “take turns” delivering food to offspring, and (if so) whether this a deliberate strategy or simply a by-product of independent care behavior. We find that babblers indeed take turns and conclude that part of the observed turn-taking is due to deliberate responsiveness, with the rest arising from the species’ breeding ecology.
Flexibility but no coordination of visits in provisioning riflemen
Khwaja, Nyil ; Preston, Stephanie A.J. ; Hatchwell, Ben J. ; Briskie, James V. ; Winney, Isabel S. ; Savage, James L. - \ 2017
Animal Behaviour 125 (2017). - ISSN 0003-3472 - p. 25 - 31.
Parental care strategies occupy a continuum from fixed investments that are consistent across contexts to flexible behaviour that largely depends on external social and environmental cues. Determining the flexibility of care behaviour is important, as it influences the outcome of investment games between multiple individuals caring for the same brood. We investigated the repeatability of provisioning behaviour and the potential for turn taking among breeders and helpers in a cooperatively breeding bird, the rifleman, Acanthisitta chloris. First, we examined whether nest visit rate is an accurate measure of investment by assessing whether carers consistently bring the same size of food, and whether food size is related to nest visit rate. Our results support the use of visit rate as a valid indicator of parental investment. Next, we calculated the repeatability of visit rate and food size to determine whether these behaviours are fixed individual traits or flexible responses to particular contexts. We found that riflemen were flexible in visit rate, supporting responsive models of care over ‘sealed bids’. Finally, we used runs tests to assess whether individual riflemen alternated visits with other carers, indicative of turn taking. We found little evidence of any such coordination of parental provisioning. We conclude that individual flexibility in parental care appears to arise through factors such as breeding status and brood demand, rather than as a real-time response to social partners.
Context-dependent effects of radio transmitter attachment on a small passerine
Snijders, Lysanne ; Nieuwe Weme, Lydia ; Goede, Piet de; Savage, James L. ; Oers, Kees van; Naguib, Marc - \ 2017
Journal of Avian Biology 48 (2017)5. - ISSN 0908-8857 - p. 650 - 659.
Biotelemetry devices provide unprecedented insights into the spatial behaviour and ecology of many animals. Quantifying the potential effects of attaching such devices to animals is essential, but certain effects may appear only in specific or particularly stressful contexts. Here we analyse the effects of radio transmitter attachment on great tits Parus major tagged over three environmentally dissimilar years, as part of a project studying social- and communication networks. When we radio-tagged birds before breeding, only those tagged in the coldest spring tended to be less likely to breed than control birds. Breeding probability was independent of relative transmitter weight (between 5 and 8% bodyweight). When we radio-tagged both parents during nestling provisioning (transmitter weight between 6 and 8%), tagged parents were more likely than control parents to desert their brood in two out of three years, while in the other year no tagged parents deserted. Tagged parents provisioning larger broods were most likely to desert, especially during lower average temperatures. Video analyses did not reveal any transmitter effects on provisioning behaviour of parents in the year with no desertion. We conclude that radio tagging before breeding did not lead to negative effects, regardless of transmitter weight, but that decisions about radio-tagging both parents during nestling provisioning need to be made with exceptional care, taking both environmental context and transmitter weight into account. Reporting results from long-term radio-tracking studies comprising several environmentally variable years is crucial to understand and predict potential transmitter effects and maximise the tremendous potential of biotelemetry. Journal of Avian Biology
Data from: Context-dependent effects of radio transmitter attachment on a small passerine
Snijders, L. ; Nieuwe Weme, L.E. ; Goede, Piet de; Savage, J.L. ; Oers, Kees van; Naguib, M. - \ 2016
biotelemetry - passerine - reproduction - Parus major
Biotelemetry devices provide unprecedented insights into the spatial behaviour and ecology of many animals. Quantifying the potential effects of attaching such devices to animals is essential, but certain effects may appear only in specific or particularly stressful contexts. Here we analyse the effects of radio transmitter attachment on great tits Parus major tagged over three environmentally dissimilar years, as part of a project studying social- and communication networks. When we radio-tagged birds before breeding, only those tagged in the coldest spring tended to be less likely to breed than control birds. Breeding probability was independent of relative transmitter weight (between 5 and 8% bodyweight). When we radio-tagged both parents during nestling provisioning (transmitter weight between 6 and 8%), tagged parents were more likely than control parents to desert their brood in two out of three years, while in the other year no tagged parents deserted. Tagged parents provisioning larger broods were most likely to desert, especially during lower average temperatures. Video analyses did not reveal any transmitter effects on provisioning behaviour of parents in the year with no desertion. We conclude that radio tagging before breeding did not lead to negative effects, regardless of transmitter weight, but that decisions about radio-tagging both parents during nestling provisioning need to be made with exceptional care, taking both environmental context and transmitter weight into account. Reporting results from long-term radio-tracking studies comprising several environmentally variable years is crucial to understand and predict potential transmitter effects and maximise the tremendous potential of biotelemetry.
Measurements of Partner Responsiveness
Savage, James - \ 2016
Introduction to Animal Behaviour
Savage, J.L. ; Snijders, L. ; Naguib, M. - \ 2016
Wageningen, The Netherlands : edX
animal welfare - wild animals - animal behaviour - dierenwelzijn - wilde dieren - diergedrag
Massive Open Online Course
Brain regions implicated in inhibitory control and appetite regulation are activated in response to food portion size and energy density in children
English, L.K. ; Fearnbach, S.N. ; Lasschuijt, M. ; Schlegel, A. ; Anderson, K. ; Harris, S. ; Fisher, J.O. ; Savage, J.S. ; Rolls, B.J. ; Keller, K.L. - \ 2016
International Journal of Obesity 40 (2016)10. - ISSN 0307-0565 - p. 1515 - 1522.

Objective:Large portions of energy-dense foods drive energy intake but the brain mechanisms underlying this effect are not clear. Our main objective was to investigate brain function in response to food images varied by portion size (PS) and energy density (ED) in children using functional magnetic resonance imaging (fMRI).Methods and design:Blood-oxygen-level-dependent (BOLD) fMRI was completed in 36 children (ages 7-10 years) after a 2-h fast while viewing food images at two levels of PS (Large PS, Small PS) and two levels of ED (High ED, Low ED). Children rated perceived fullness pre- and post-fMRI, as well as liking of images on visual analog scales post-fMRI. Anthropometrics were completed 4 weeks before the fMRI. Large PS vs Small PS and High ED vs Low ED were compared with region-of-interest analyses using Brain Voyager v 2.8.Results:Region-of-interest analyses revealed that activation in the right inferior frontal gyrus (P=0.03) was greater for Large PS vs Small PS. Activation was reduced for High ED vs Low ED in the left hypothalamus (P=0.03). Main effects were no longer significant after adjustment for pre-fMRI fullness and liking ratings (PS, P=0.92; ED, P=0.58).Conclusion:This is the first fMRI study to report increased activation to large portions in a brain region that is involved in inhibitory control. These findings may contribute to understanding why some children overeat when presented with large portions of palatable food.

Diversity and function of vocalisations in the cooperatively breeding Chestnut-crowned Babbler
Crane, Jodie M.S. ; Savage, James L. ; Russell, Andrew F. - \ 2016
Emu 116 (2016)3. - ISSN 0158-4197 - p. 241 - 253.
avian acoustics - Pomatostomidae - social brain hypothesis - social complexity hypothesis.

Vocalisations represent the primary mode of communication for most birds and vary greatly in form and function within and between species. Cataloguing the vocal repertoire of a species is a key foundation for behavioural research, as it provides both an objective measure of vocal complexity and a basis for further studies of vocal function. Here we present a descriptive catalogue of the vocal repertoire of the Chestnut-crowned Babbler (Pomatostomus ruficeps), a cooperatively breeding passerine endemic to inland south-eastern Australia. Using behavioural observations and simple methods of acoustic classification we identify and suggest functions for 13 main types of vocalisations, and also report five less common vocalisations that could not be assigned a unique function. Babblers possess no song, in the sense of a vocalisation primarily used for inter-group or territorial communication. The 13 calls with clear functions can be broadly classified into three alarm calls, five contact calls, and four social-interaction calls, with a final call used in both social and alarm contexts. This study represents the first catalogue of vocal repertoire in pomatostomid babblers, and aims to contribute to future comparative analyses of vocal complexity and inspire further work on the relationship between call structure and function.

Brain response to images of food varying in energy density is associated with body composition in 7- to 10-year-old children : Results of an exploratory study
Fearnbach, S.N. ; English, Laural K. ; Lasschuijt, Marlou ; Wilson, Stephen J. ; Savage, Jennifer S. ; Fisher, Jennifer O. ; Rolls, Barbara J. ; Keller, Kathleen L. - \ 2016
Physiology and Behavior 162 (2016). - ISSN 0031-9384 - p. 3 - 9.
Body composition - Children - Energy density - Fat-free mass - fMRI - Neuroimaging

Energy balance is regulated by a multifaceted system of physiological signals that influence energy intake and expenditure. Therefore, variability in the brain's response to food may be partially explained by differences in levels of metabolically active tissues throughout the body, including fat-free mass (FFM) and fat mass (FM). The purpose of this study was to test the hypothesis that children's body composition would be related to their brain response to food images varying in energy density (ED), a measure of energy content per weight of food. Functional magnetic resonance imaging (fMRI) was used to measure brain response to High (> 1.5 kcal/g) and Low (

Turn-taking in avian parental care
Savage, James - \ 2015
How do we analyse parental care on a visit-by-visit basis?
Savage, James - \ 2015
invited talk
How do social associations over winter influence breeding behaviour in the great tit?
Savage, James - \ 2015
Unrelated helpers neither signal contributions nor suffer retribution in chestnut-crowed babblers
Nomano, F.Y. ; Browning, L.E. ; Savage, J.L. ; Rollins, L.A. ; Griffith, S.C. ; Russell, A.F. - \ 2015
Behavioral Ecology 26 (2015)4. - ISSN 1045-2249 - p. 986 - 995.
cooperatively breeding bird - pay-to-stay - crowned babbler - pomatostomus-ruficeps - group augmentation - helping-behavior - predation risk - evolution - coefficients - relatedness
Alloparental care by distant/nonkin that accrue few kin-selected benefits requires direct fitness benefits to evolve. The pay-to-stay hypothesis, under which helpers contribute to alloparental care to avoid being expelled from the group by dominant individuals, offers one such explanation. Here, we investigated 2 key predictions derived from the pay-to-stay hypothesis using the chestnut-crowed babbler, Pomatostomus ruficeps, a cooperatively breeding bird where helping by distant/nonkin is common (18% of nonbreeding helpers). First, we found no indication that distant or nonkin male helpers advertised their contributions toward the primary male breeder. Helpers unrelated to both breeders were unresponsive to provisioning rates of the dominant male, whereas helpers that were related to either the breeding male or to both members of the pair were responsive. In addition, unrelated male helpers did not advertise their contributions to provisioning by disproportionately synchronizing their provisioning events with those of the primary male breeder or by provisioning nestlings immediately after him. Second, no helper, irrespective of its relatedness to the dominant breeders, received aggression when released back into the group following temporary removal for 1–2 days. We therefore find no compelling support for the hypothesis that pay-to-stay mechanisms account for the cooperative behavior of unrelated males in chestnut-crowned babblers.
Experimental Evidence for Phonemic Contrasts in a Nonhuman Vocal System
Engesser, S. ; Crane, J.M.S. ; Savage, J.L. ; Russell, A.F. ; Townsend, S.W. - \ 2015
PloS Biology 13 (2015)6. - ISSN 1545-7885
phonological awareness - language - evolution - song - vocalizations - syntax - roles - calls - bird - communication
The ability to generate new meaning by rearranging combinations of meaningless sounds is a fundamental component of language. Although animal vocalizations often comprise combinations of meaningless acoustic elements, evidence that rearranging such combinations generates functionally distinct meaning is lacking. Here, we provide evidence for this basic ability in calls of the chestnut-crowned babbler (Pomatostomus ruficeps), a highly cooperative bird of the Australian arid zone. Using acoustic analyses, natural observations, and a series of controlled playback experiments, we demonstrate that this species uses the same acoustic elements (A and B) in different arrangements (AB or BAB) to create two functionally distinct vocalizations. Specifically, the addition or omission of a contextually meaningless acoustic element at a single position generates a phoneme-like contrast that is sufficient to distinguish the meaning between the two calls. Our results indicate that the capacity to rearrange meaningless sounds in order to create new signals occurs outside of humans. We suggest that phonemic contrasts represent a rudimentary form of phoneme structure and a potential early step towards the generative phonemic system of human language.
Maternal allocation in cooperative breeders: Should mothers match or compensate for expected helper contributions?
Savage, J.L. ; Russell, A.F. ; Johnstone, R.A. - \ 2015
Animal Behaviour 102 (2015). - ISSN 0003-3472 - p. 189 - 197.
egg size - clutch size - trade-off - parental investment - male attractiveness - offspring quality - breeding birds - body-size - number - growth
Among species with variable numbers of individuals contributing to offspring care, an individual's investment strategy should depend upon both the size of the breeding group and the relative contributions of each carer. Existing theoretical work on carer investment rules has, however, largely focused on biparental care, and on modelling offspring provisioning in isolation from other stages of investment. Consequently, there has been little exploration of how maternal investment prior to birth might be expected to influence carer provisioning decisions after birth, and how these should be modified by the number of carers present. In particular, it is unclear whether mothers should increase or decrease their investment in each offspring under favourable rearing conditions, and whether this differs under alternative assumptions about the consequences of being ‘high quality’ at birth. We develop a game-theoretical model of cooperative care that incorporates female control of prebirth investment, and allow increased maternal investment to either substitute for later investment (giving offspring a ‘head start’) or raise the value of later investment (a ‘silver spoon’). We show that mothers reduce prebirth investment under better rearing conditions (more helpers) when investment is substitutable, leading to concealed helper effects. In contrast, when maternal prebirth investment primes offspring to benefit more from postbirth care, mothers should take advantage of good care environments by investing more in offspring both before and after birth. These results provide novel mechanisms to explain contrasting patterns of maternal investment across cooperative breeders.
Using ex ante output elicitation to model state-contingent technologies
Chambers, R.G. ; Serra, T. ; Stefanou, S.E. - \ 2015
Journal of Productivity Analysis 43 (2015)1. - ISSN 0895-562X - p. 75 - 83.
technical efficiency - cheap talk - cost - distributions - uncertainty - economics
Survey-elicited ex ante outputs are used to develop an empirical representation of an Arrow–Debreu–Savage state-contingent technology in an activity-analysis framework. An empirical test of output-cubicality is developed for that framework. We apply those tools to assess production characteristics of a sample of Catalan farmers specialized in arable crops. Results suggest that imposing nonsubstitutability between ex ante outputs results in no significant loss of information. Even though the technology appears to be output cubical, efficiency measurements based on ex post output observations do not appear to adequately represent the stochastic production environment and apparently yield downward biased technical efficiency measures.
Information Needs of Family Carers of People with Diabetes at the End of Life: A Literature Review
Dikkers, M.F. ; Dunning, T. ; Savage, S. - \ 2013
Journal of Palliative Medicine 16 (2013)12. - ISSN 1096-6218 - p. 1617 - 1623.
palliative care - advanced cancer - management - caregivers
Background: Recent research identified the issue that family carers of people with diabetes at the end of life (EOL) did not receive sufficient information to enable them to help their relative manage their diabetes at the EOL. Aim: The aim of the current study was to identify by conducting a literature review the information needs of family carers of people with diabetes at the EOL by conducting a literature review. Method: A comprehensive review of the literature was conducted by searching the following databases: CINAHL, PubMed, PsychInfo, Scopus, and SocINDEX. The grey literature was also searched for papers relevant to the aim. All study designs were included. A content analysis of relevant papers was undertaken to identify themes. Results: Sixteen of the more than 300 papers identified addressed the information needs of family carers of people with diabetes at the EOL and were included in the review. Five key themes were identified from the papers reviewed: (1) performing diabetes care tasks, (2) focus of care, (3) blood glucose management, (4) EOL stages, and (5) involving patients and family carers in decisions about diabetes care. Most of the 16 papers represented the views of health professionals and focused on the need to provide information about the medical aspects of diabetes management. Conclusions: The review suggests further research is needed to identify the information needs of family carers of people with diabetes at the EOL to enable interventions to be developed to support the family carers and meet their unique information needs.
Unravelling hazards of nanoparticles to earthworms, from gene to population
Ploeg, M. van der - \ 2013
University. Promotor(en): Ivonne Rietjens, co-promotor(en): Nico van den Brink. - S.l. : s.n. - ISBN 9789461734440 - 192
aardwormen - lumbricus rubellus - nanotechnologie - blootstelling - ecotoxicologie - earthworms - nanotechnology - exposure - ecotoxicology

Nanotechnology is an expeditiously growing field, where engineered nanoparticles are being incorporated in many different applications, from food to waste water treatment (Dekkers et al. 2011; Gottschalk and Nowack 2011; Savage and Diallo 2005). Due to this large scale production and use of nanoparticles, their release into the environment seems inevitable (Crane et al. 2008; Handy et al. 2008a; Oberdörster et al. 2005). Actual exposure levels of nanoparticles under field conditions and the hazards of nanoparticle exposure to the environment are poorly understood, especially for the soil environment (Kahru and Dubourguier 2010; Navarro et al. 2008; Shoults-Wilson et al. 2011a).

Given the need for better characterization of hazards of engineered nanoparticles to the environment and soil organisms in particular, the aim of the present thesis was to investigate effects of nanoparticle exposure on the earthworm Lumbricus rubellus, as a model organism for soil ecotoxicology, and to contribute to the development of effect markers for engineered nanoparticle exposure in this model.

The present thesis was divided in different chapters. Chapter 1 provides an introduction to the topic and discusses the importance of research on the hazards of exposure to engineered nanoparticles. Furthermore, the aim and outline of the thesis are presented, with background information on the model organism, effect markers and nanoparticles.

In chapter 2 effects of exposure to the fullerene C60 (nominal concentrations 0, 15.4 and 154 mg C60/kg soil) on survival and growth during the different life stages of L. rubellus (cocoon, juvenile, subadult and adult), as well as reproduction were quantified. These important individual endpoints for population dynamics were incorporated in a continuous-time life-history model (Baveco and De Roos 1996; De Roos 2008). In this way, effects of C60 exposure on the individual endpoints could be extrapolated to implications for population growth rate and life stage distribution, i.e. the development of the population in terms of number of individuals in the different life stages. These implications at the population level may be more relevant for the ecological impact of C60 than effects on endpoints at the individual level (Klok et al. 2006; Widarto et al. 2004). At the individual level C60 exposure caused significant adverse effects on cocoon production, juvenile growth rate and survival. When these endpoints were used to model effects on the population level, reduced population growth rates with increasing C60 concentrations were observed. Furthermore, a shift in life stage structure was shown for C60 exposed populations, towards a larger proportion of juveniles. This result implies that the lower juvenile growth rate induced by C60 exposure resulted in a larger proportion of juveniles, despite increased mortality among juveniles. Overall, this study implied serious consequences of C60 exposure for L. rubellus earthworm populations, even at the lowest level of exposure tested. Furthermore, it showed that juveniles were more sensitive to C60 exposure than adults.

To complement the observations made on survival, growth and reproduction described in chapter 2, subsequent investigations on cellular and molecular responses of the earthworms to C60 exposure were performed (chapter 3). A set of established effect markers was used, which reflect different levels of biological organisation in the earthworm and may inform on the toxic mechanisms of adverse effects induced by C60 exposure (Handy et al. 2002; Heckmann et al. 2008). At the molecular level, four specific effect markers were selected, including markers for generic stress (heat shock protein 70 (HSP70) (van Straalen and Roelofs 2006), for oxidative stress (catalase and glutathione-S-transferase (GST) (Kohen and Nyska 2002) and for an immune response (coelomic cytolytic factor-1 (CCF-1) (Olivares Fontt et al. 2002). At the tissue level, histological analyses were used to identify damage to cells and tissues, and indications of inflammation in the tissues. In these investigations, exposure to C60 (0, 15 or 154 mg C60/kg soil) affected gene expression of HSP70 significantly. Gene expression of CCF-1 did not alter in adult earthworms exposed for four weeks, but was significantly down-regulated after lifelong exposure (from cocoon stage to adulthood) of earthworms, already to the lowest C60 exposure level. No significant trends were noted for catalase and glutathione-S-transferase (GST) gene expression or enzyme activity. Tissue samples of the C60 exposed earthworms from both experiments and exposure levels, showed a damaged cuticle with underlying pathologies of epidermis and muscles. Additionally, the gut barrier was not fully intact. However, tissue repair was also observed in these earthworms. In conclusion, this study demonstrated effects of sub-lethal C60 exposure on L. rubellus earthworms, at the level of gene expression and tissue integrity.

Although tissue injury is generally associated with an inflammatory response, as part of tissue repair (Cikutovic et al. 1999; Goven et al. 1994), the tissue damage observed for the in vivo C60 exposed earthworms in chapter 3 appeareded to occur without accompanying induced immune responses. The CCF-1 gene expression level was reduced in the C60 exposed earthworms, and histological observations did not show infiltration of damaged tissues by immune cells. In order to obtain further insight in mechanisms of effects observed at the molecular and tissue level on immune related parameters, the sensitivity of immune cells (coelomocytes) of L. rubellus earthworms towards exposure to selected nanoparticles was investigated in vitro (chapter 4). To this end, coelomocytes were isolated from unexposed adult L. rubellus earthworms and exposed to C60 in vitro. After exposure, these coelomocytes were tested for cellular viability, phagocytic activity and CCF-1 gene expression levels. The gene expression of CCF-1 was most affected, demonstrating a strong reduction, which indicated immunosuppression. Experiments with NR8383 rat macrophage cells and tri-block copolymer nanoparticles were used to compare sensitivity of the cell types and showed the usefulness of coelomocytes as a test system for nano-immunotoxicity in general. Overall, this study indicated that the absence of an immune response, in case of tissue injuries observed after in vivo C60 exposure, is likely caused by immunosuppression rather than coelomocyte mortality.

In subsequent investigations, the experiments performed for C60 were also carried out with silver nanoparticles (AgNP), both in vivo and in vitro (chapter 5). Effects of AgNP were assessed in vivo at nominal concentrations of 0, 1.5 (low), 15.4 (medium) and 154 (high) mg Ag/kg soil and compared to effects of silver ions, added as AgNO3 (nominal concentration 15.4 mg Ag/kg soil). In a four week reproduction assay, the high AgNP and AgNO3 treatments had a significant effect on cocoon production and high AgNP exposure also caused a reduction in weight gain of the adult earthworms. No juveniles survived the high AgNP treatment, therefore only F1 earthworms from the other exposure treatments were monitored for survival and growth, until adulthood. These individual endpoints were used to model effects on the population level. The low and medium AgNP as well as the AgNO3 treatments significantly reduced the population growth rate. The high AgNP treatment caused complete failure of the population growth. Furthermore, histological examination of the earthworms from all AgNP exposure treatments demonstrated tissue damage, with injuries mainly at the external barriers, e.g. the cuticle and the gut epithelium. In addition, effects of AgNP exposure were assessed in vitro and a reduction of coelomocyte viability was observed in a concentration-dependent manner, although the EC50 was fourteen times higher compared with that for Ag ions, added as AgNO3. Furthermore, characterisation of the in vivo exposure media implied that AgNP remained present in the soil in single and aggregated state, releasing Ag to the soil pore water up to at least eleven months. The ionic fraction of Ag in soils has been suggested to be bioavailable to organisms and (largely) responsible for the observed AgNP toxicity (Coutris et al. 2012; Koo, et al. 2011; Shoults-Wilson et al. 2011b). In comparison, the AgNO3 seemed to dissolve rapidly, as is also known for this metal salt, and fixation of Ag ions by the soil presumably led to a quick reduction of Ag bioavailability (Atkins and Jones 2000; Coutris et al. 2012; Ratte 1999). This is in line with the observation that effects were more prolonged in the AgNP treatments in comparison with the AgNO3 exposed animals. In conclusion, this study indicated that AgNP exposure may seriously affect earthworm populations, with the ability to cause immunotoxicity, injury to the external barriers of the earthworm body and a reduction in growth, reproduction and juvenile survival.

Finally, chapter 6 presents a discussion on the findings of the present thesis and provides suggestions for future research.

Enhanced NMR-based profiling of polyphenols in commercially available grape juices using solid-phase extraction
Savage, A.K. ; Duynhoven, J.P.M. van; Tucker, G. ; Daykin, C. - \ 2011
Magnetic Resonance in Chemistry 49 (2011)1. - ISSN 0749-1581 - p. S27 - S36.
nuclear-magnetic-resonance - fruit juices - h-1-nmr spectroscopy - liquid-extraction - by-products - green tea - wine - quality - identification - chromatography
Grapes and related products, such as juices, and in particular, their polyphenols, have previously been associated with many health benefits, such as protection against cardiovascular disease. Within grapes, a large range of structurally diverse polyphenols can be present, and their characterisation stands as a challenge. 1H NMR spectroscopy in principle would provide a rapid, nondestructive and straightforward method for profiling of polyphenols. However, polyphenol profiling and identification in grape juices is hindered because of signals of prevailing carbohydrates causing spectral overlap and compromising dynamic range. This study describes the development of an extraction method prior to analysis using 1H NMR spectroscopy, which can, potentially, significantly increase the number of detectable polyphenols and aid their identification, by reduction of signal overlap and selective removal of heavily dominating compounds such as sugars.
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